Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter.
A membrane protein of relative molecular mass (Mr) 127,000 was identified by photoaffinity labelling as (a component of) the uptake system for small peptides and beta-lactam antibiotics in rabbit small intestine. This binding protein is a microheterogeneous glycosylated integral membrane protein which could be solubilized with non-ionic detergents and enriched by lectin affinity chromatography on wheat germ lectin agarose. For the final purification of this protein and separation from aminopeptidase N of Mr 127,000, fast protein liquid chromatography (FPLC) was used. Gel permeation, hydroxyapatite and hydrophobic interaction chromatography were not successful for the purification of the 127,000-dalton binding protein. By anion-exchange chromatography on a Mono Q column with either Triton X-100 or n-octylglucoside as detergent, a partial separation of the 127,000-dalton binding protein from aminopeptidase N was achieved. By cation-exchange chromatography on a Mono S HR 5/5 column at pH 4.5 using Triton X-100 as detergent also only a partial separation from aminopeptidase N could be achieved. If, however, Triton X-100 was replaced with n-octylglucoside, the binding protein for beta-lactam antibiotics and small peptides of Mr 127,000 could be completely separated from aminopeptidase N. These results indicate that Triton X-100 should be avoided for the purification of integral membrane proteins because mixed protein-detergent micelles of high molecular weight prevent a separation into the individual membrane proteins. The putative peptide transport protein was finally purified by rechromatography on Mono S and was obtained more than 95% pure as determined densitometrically after sodium dodecyl sulphate gel electrophoresis. By application of FPLC even microheterogeneous membrane glycoproteins from the intestinal mucosa can be purified to such an extent that a sequence analysis and immunohistochemical localization with antibodies prepared from the purified protein is possible.
Kramer
W
W
Hoechst Aktiengesellschaft, Postfach 800320, Frankfurt am Main, F.R.G.
Girbig
F
F
Gutjahr
U
U
Leipe
I
I
D000345:Affinity Labels; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D007421:Intestine, Small / Q000737:chemistry*; D008871:Microvilli / Q000737:chemistry; D008970:Molecular Weight; D010400:Penicillin G / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011817:Rabbits
D000345:Affinity Labels; D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D006023:Glycoproteins; D010455:Peptides; D010400:Penicillin G